Methods and devices for analysis of defined multicellular combinations

ABSTRACT

Methods for cell analysis are provided, comprising cell capturing, characterization, transport, and culture. In an exemplary method individual cells (and/or cellular units) are flowed into a microfluidic channel, the channel is partitioned into a plurality of contiguous segments, capturing at least one cell in at least one segment. A characteristic of one or more captured cells is determined and the cell(s) and combinations of cells are transported to specified cell holding chamber(s) based on the determined characteristic(s). Also provided are devices and systems for cell analysis.

RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.14/775,685, filed Mar. 14, 2014, entitled “METHODS AND DEVICES FORANALYSIS OF DEFINED MULTICELLULAR COMBINATIONS”, which is a US NationalPhase of PCT Application No. PCT/US2014/029344 filed on Mar. 14, 2014,which claims priority to U.S. provisional application No. 61/852,135,filed Mar. 15, 2013, the content of which is incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to methods and devices for analysis ofindividual cells and defined combinations of cells.

BACKGROUND

A number of publications have discussed methods for manipulation ofsingle cells, including the following (none of which is admitted to beprior art): Sims et al., 2007, “Analysis of single mammalian cellson-chip” Lab Chip 7:423-440; Wheeler et al., 2003, “Microfluidic devicefor single-cell analysis” Anal Chem 75:3581-3586; Skelley et al., 2009“Microfluidic control of cell pairing and fusion” Nat Methods 6:147-152;Marcus et al., 2006, “Microfluidic single-cell mRNA isolation andanalysis” Anal Chem 78:3084-3089; Bontoux et al., 2008 “Integratingwhole transcriptome assays on a lab-on-a-chip for single cell geneprofiling” Lab Chip 8:443-450; Zhong et al., 2008 “A microfluidicprocessor for gene expression profiling of single human embryonic stemcells” Lab Chip 8:68-74; Wheeler 2003 “Microfluidic Device forSingle-Cell Analysis Anal. Chem.” 75:3581-3586; and White et al., Aug.23, 2011 “High-throughput microfluidic single-cell RT-qPCR PNAS” Vol.108, 34:13999-14004. Each of the aforelisted publications isincorporated herein by reference.

The present invention provides a new method with features and advantagesnot found in prior art methods and devices.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to methods and devices for analysis ofindividual cells and defined combinations of cells. The followingembodiments are not intended to be limiting. Rather, it is understoodthat the examples and embodiments described herein are for illustrativepurposes only and that various modifications or changes in light thereofwill be suggested to persons skilled in the art and are to be includedwithin the spirit and purview of this application

In the following recitation of embodiments and throughout the entirespecification, for simplicity, reference generally is made to an“individual cell” or a “single captured cell.” For each and every suchrecitation, except where otherwise clear from context, it iscontemplated that in alternative embodiments “individual cell” and a“single captured cell” should be read as “individual cellular unit” and“single captured cellular unit.” Also contemplated are embodiments inwhich single cells are captured in some segments and process and singlecell units are captured in other segments and processed.

In one aspect, the application discloses Aspect 1, which is a method forcell analysis, comprising carrying out at least two rounds of cellcapturing, characterization, and transport, each round comprising: a)flowing a solution comprising a plurality of individual cells (and/orcellular units) into a first microfluidic channel; b) partitioning thechannel into a plurality of contiguous segments (S), thereby capturingat least one cell in at least one segment, wherein i) one or more ofsaid segments comprises a single captured cell (and/or single capturedcellular unit), and c) determining at least one characteristic of one ormore of said single captured cells and/or cellular units; and d)independently transporting each said captured cell or unit to aspecified cell holding chamber based on the determined characteristic,whereby for each specified destination chamber the characteristic(s) ofcell(s) transported thereto is known.

In one aspect, the application discloses Aspect 2, the method of aspect1 wherein at least three, at least four, at least five, at least six, atleast 10, at least 15, or at least 20 rounds of cell capturing,characterization, and transport are carried out.

In one aspect, the application discloses Aspect 3, the method of anypreceding aspect wherein in (b) at least 30% of said segments compriseno more than one cell.

In one aspect, the application discloses Aspect 4, the method of aspect3 wherein the majority of said segments comprise no more than one cell.

In one aspect, the application discloses Aspect 5, the method of anypreceding aspect wherein one or more segments does not comprise anycell.

In one aspect, the application discloses Aspect 6, the method of anypreceding aspect wherein the number of individual cells (or cellularunits) flowed into the portion of the first microfluidic channel that ispartitioned is less than the number of segments produced as a result ofthe partition.

In one aspect, the application discloses Aspect 7, the method of anypreceding aspect wherein method is carried out in a device comprisingtwo or more partitioning channels and a cell may be transported from anysegment of any partitioning channel to any cell holding chamber.

In one aspect, the application discloses Aspect 8, the method of aspect7 wherein there are two partitioning channels.

In one aspect, the application discloses Aspect 9, the method of anypreceding aspect wherein the lumen of the first (partitioning)microfluidic channel is substantially featureless.

In one aspect, the application discloses Aspect 10, the method of any ofaspects 1-9 wherein the characteristic determined is cell size,morphology, or the presence or absence of an extracellular orintracellular antigen.

In one aspect, the application discloses Aspect 11, the method of any ofaspects 1-9 wherein the characteristic determined is a cell behavior.

In one aspect, the application discloses Aspect 12, the method of any ofaspects 1-9 wherein the characteristic determined the response by thecell to a physical, chemical or biological challenge.

In one aspect, the application discloses Aspect 13, the method of anypreceding aspect wherein said cells are transported by bulk fluid flow.

In one aspect, the application discloses Aspect 14, the method of anypreceding aspect wherein a compound manifold system is used to transportcells from any segment to any cell holding chamber.

In one aspect, the application discloses Aspect 15, the method of anypreceding aspect wherein each cell transported from a segment to a cellholding chamber is transported though (e.g., flows though) the sameconnector channel.

In one aspect, the application discloses Aspect 16, the method of anypreceding aspect wherein each of said segments is in fluidiccommunication with a common second microfluidic channel, and saidcaptured cells are transported through the second microfluidic channelin transit to a specified cell holding chamber.

In one aspect, the application discloses Aspect 17, the method of anypreceding aspect in which at least 10 individual cells (or cellularunits) are transported from segments to cell holding chambers and/or atleast 10 cell holding chambers are occupied by cells.

In one aspect, the application discloses Aspect 18, the method of aspect16 or 17 in which the compound manifold connects at least 5 segments andat least 5 cell holding chambers.

In one aspect, the application discloses Aspect 19, the method of aspect18 in which the compound manifold connects at least 10 segments and atleast 10 cell holding chambers.

In one aspect, the application discloses Aspect 20, the method of aspect19 in which the compound manifold connects at least 10 segments and atleast 10 cell holding chambers.

In one aspect, the application discloses Aspect 21, the method of aspect20 in which the compound manifold connects at least 20 segments and atleast 20 cell holding chambers.

In one aspect, the application discloses Aspect 22, the method of aspect21 in which the compound manifold connects at least 10 segments and atleast 10 to 100 cell holding chambers.

In one aspect, the application discloses Aspect 23, the method of aspect22 in which the compound manifold connects at least 50 segments and atleast 50-100 cell holding chambers.

In one aspect, the application discloses Aspect 24, the method of anypreceding aspect wherein the ratio of the number of segments to thenumber of cell holding chambers is greater than 1.

In one aspect, the application discloses Aspect 25, the method of aspect24 wherein the number of segments is greater than the number of cellholding chambers by a factor of about 10%, about 20%, about 50% about75% about 100% or about 200%.

In one aspect, the application discloses Aspect 26, the method of anypreceding aspect in which multiple cells are individually transported tothe same cell holding chamber, thereby producing a cell holding chambercomprising a defined combination of cells.

In one aspect, the application discloses Aspect 27, the method of aspect26 in which two, three or four cells are individually transported to thesame cell holding chamber.

In one aspect, the application discloses Aspect 28, the method of aspect27 in which each individually transported cell is captured in adifferent round of partitioning.

In one aspect, the application discloses Aspect 29, the method of anypreceding aspect wherein the cells flowed into the first microfluidicchannel are eukaryotic cells.

In one aspect, the application discloses Aspect 30, the method of aspect29 wherein the cells are animal cells.

In one aspect, the application discloses Aspect 31, the method of aspect30 wherein the cells are human.

In one aspect, the application discloses Aspect 32, the method of anypreceding aspect wherein the cells are plant cells.

In one aspect, the application discloses Aspect 33, the method of anypreceding aspect wherein the solution comprising a plurality ofindividual cells (or cellular units) in (a) comprises cells from twodifferent eukaryotic species.

In one aspect, the application discloses Aspect 34, the method of anypreceding aspect wherein the solution comprising a plurality ofindividual cells (or cellular units) in (a) comprises cells from twodifferent individuals or specimens of the same species.

In one aspect, the application discloses Aspect 35, the method of anypreceding aspect, wherein the plurality of individual cells (or cellularunits) in (a) comprises rare cells and other cells, at least one cellthat is transported in step (d) to a cell holding chamber is a rarecell, and the ratio in said solution of said other cells to said rarecells is greater than 100:1, sometimes greater than 1000:1, sometimesgreater than 10,000:1, and sometimes greater than 100,000:1.

In one aspect, the application discloses Aspect 36, the method of aspect35 wherein the rare cell is a stem cell, a tumor cell, optionally acirculating tumor cell, a circulating endothelial cell, or a fetal cell.

In one aspect, the application discloses Aspect 37, the method of any ofaspects 1-36 in which cells are cultured in the cell holding chamber.

In one aspect, the application discloses Aspect 38, the method of aspect37 in which the cells are cultured for from about 1 hour to about 4weeks.

In one aspect, the application discloses Aspect 39, the method of aspect37 in which the cells are cultured for from about 1 hour to about 24hours.

In one aspect, the application discloses Aspect 40, the method of any ofaspects 37-39 in which the cells divide to produce progeny.

In one aspect, the application discloses Aspect 41, the method of any ofaspects 37-40 in which the cells are observed during culture.

In one aspect, the application discloses Aspect 42, the method of any ofaspects 37-41 in which the cells are challenged during culture.

In one aspect, the application discloses Aspect 43, the method of any ofaspects 37-42 in which the challenge comprises exposing the cells to anagent

In one aspect, the application discloses Aspect 44, the method of aspect43 in which the agent is a drug, test agent, protein, nucleic acid orsmall molecule.

In one aspect, the application discloses Aspect 45, the method of any ofaspects 37-44 in which a first cell or combination of cells is culturedfor at least one hour, and then one or more additional cells obtained bycell capturing, characterization, and transport in the device isintroduced into the cell holding chamber.

In one aspect, the application discloses Aspect 46, the method of any ofaspects 37-45 wherein after a period of culture, viable cells areharvested from a cell holding chamber.

In one aspect, the application discloses Aspect 47, the method of any ofaspects 37-46 wherein after a period of culture, cells in a cell holdingchamber are fixed in situ.

In one aspect, the application discloses Aspect 48, the method of any ofaspects 37-45 in which a reagent, solution or physical stimulus isapplied to a cell or cells in one or more cell holding chambers, andresults in lysis of said cell or cells.

In one aspect, the application discloses Aspect 49, the method of aspect48 wherein macromolecules released from the lysed cells are transportedout of at least one cell holding chamber.

In one aspect, the application discloses Aspect 50, the method of aspect49 wherein macromolecules are collected.

In one aspect, the application discloses Aspect 51, the method of aspect49 wherein macromolecules released from the lysed cells are transportedout of a the cell holding chamber into a corresponding microfluidicchamber, wherein the corresponding microfluidic chamber is in fluidiccommunication with said cell holding chamber and not with other cellholding chambers.

In one aspect, the application discloses Aspect 52, the method of aspect50 or 51 wherein the macromolecules are nucleic acids, optionally thenucleic acids are amplified (optionally reverse transcribed), andoptionally the nucleic acids or corresponding amplicons are assayed inthe microfluidic system wherein said steps occur all occur within thefluidic circuit, where optionally the assay determined a geneticcharacteristic or gene, RNA or protein expression pattern.

In one aspect, the application discloses Aspect 53, a chamber in amicrofluidic device that is configured for receiving and maintaininglive cells, the chamber comprising: an input channel configured so thatnucleated eukaryotic cells can pass intact through the input channel andinto the chamber; a plurality of four or more drain channels configuredso that fluid but not nucleated eukaryotic cells can exit the chamber;and a gas permeable membrane separating the chamber from a supplychannel, wherein the supply channel is configured to bring a gaseousmixture to the chamber, and the gas permeable membrane is configured sothat gasses may be exchanged between the chamber and the supply channel.

In one aspect, the application discloses Aspect 54, the chamber ofaspect 53, wherein the chamber or the input channel is connected to areagent channel configured for supplying a fluid comprising one or morereagents for maintaining or treating cells in the chamber.

In one aspect, the application discloses Aspect 55, the chamber ofaspect 54, wherein the reagent channel is connected to a supply of afluid containing cell nutrients.

In one aspect, the application discloses Aspect 56, the chamber ofaspect 54, wherein the reagent channel is connected to a supply of afluid that lyses cells upon delivery of the fluid into the chamber.

In one aspect, the application discloses Aspect 57, the chamber of anyof aspects 53-56, wherein the input channel tapers towards the chamber,thereby directing cells towards the chamber's center.

In one aspect, the application discloses Aspect 58, the chamber of anyof aspects 53-57, wherein the drain channels are distributed around thechamber in a manner that allows fluid to flow from the input channelinto the chamber and out the drain channels without drawing cells in thechamber towards the drain channels.

In one aspect, the application discloses Aspect 59, the chamber ofaspect 58, wherein the chamber has a perimeter that is substantiallycircular or oval in shape, and the drain channels are distributed overmore than 180 degrees of the perimeter.

In one aspect, the application discloses Aspect 60, the chamber of anyof aspects 53-59, comprising ten or more drain channels.

In one aspect, the application discloses Aspect 61, the chamber of anyof aspects 53-60, wherein the drain channels connect to the chamberthrough drain openings and the input channel connects to the channelthough an input opening, and the diameter of the drain openings is lessthan 20% of the diameter of the input opening, thereby inhibitingpassage of intact cells into the drain channels.

In one aspect, the application discloses Aspect 62, the chamber of anyof aspects 53-61, wherein the drain channels contain beads or a filter,thereby inhibiting passage of intact cells into the drain channels.

In one aspect, the application discloses Aspect 63, the chamber of anyof aspects 53-62, large enough to accommodate at least 3 eukaryoticcells.

In one aspect, the application discloses Aspect 64, the chamber of anyof aspects 53-63, large enough to accommodate at least 10 eukaryoticcells.

In one aspect, the application discloses Aspect 65, the chamber of anyof aspects 53-64, at least 50 microns across at its narrowest diameter.

In one aspect, the application discloses Aspect 66, the chamber of anyof aspects 53-65, at least 200 microns across at its narrowest diameter.

In one aspect, the application discloses Aspect 67, the chamber of anyof aspects 53-66, comprising a convex lower surface.

In one aspect, the application discloses Aspect 68, the chamber of anyof aspects 53-67, comprising a lower surface coated with a substancethat promotes cell adhesion.

In one aspect, the application discloses Aspect 69, the chamber ofaspect 68, wherein the substance is an extracellular matrix.

In one aspect, the application discloses Aspect 70, the chamber ofaspect 68, wherein the substance is fibronectin.

In one aspect, the application discloses Aspect 71, the chamber of anyof aspects 53-70, wherein the chamber, the input channel, and the drainchannels are filled with fluid.

In one aspect, the application discloses Aspect 72, the chamber of anyof aspects 53-71, containing at least two eukaryotic cells.

In one aspect, the application discloses Aspect 73, a device comprisinga plurality of four or more chambers according to any of aspects 53-71,each having its own separate input channel, wherein each of the inputchannels is configured to supply cells from a common source.

In one aspect, the application discloses Aspect 74, a device accordingto aspect 73, wherein the separate input channels each has a valve thatcan be operated independently of valves in the other input channels.

In one aspect, the application discloses Aspect 75, a device accordingto aspect 73 or 74, wherein the separate input channels are part of afirst multiplexer, and the common source is a connection channel thatconnects the first multiplexer to a second multiplexer, wherein thesecond multiplexer is configured to deliver cells from a plurality ofdifferent sources to the connection channel.

In one aspect, the application discloses Aspect 76, a device accordingto aspect 75, wherein the different sources are partitioned regions of acommon partitioning channel.

In one aspect, the application discloses Aspect 77, a method ofmaintaining cells in culture, comprising delivering cells to a chamberaccording to any of aspects 53-72, supplying nutrient medium into thechamber through the input channel or a separate reagent channel, andsupplying gas to the gas permeable membrane through the supply channel.

In one aspect, the application discloses Aspect 78, the method of aspect77, further comprising individually selecting the cells delivered to thechamber from a cell mixture.

In one aspect, the application discloses Aspect 79, the method ofextracting intracellular components from one or more cells that arepresent in a chamber according to any of aspects 53-72, comprisinglysing the cells by delivering a fluid that causes cell lysis into thechamber, and retrieving products of the lysis from the chamber.

In one aspect, the application discloses Aspect 80, the method of aspect79, wherein the products are retrieved through the drain channels.

In one aspect, the application discloses Aspect 81, the method of aspect79 or 80, wherein the products comprise nucleic acid.

In one aspect, the application discloses Aspect 82, the method of any ofaspects 78-81, further comprising subjecting the products to a chemicalor biochemical reaction.

In one aspect, the application discloses Aspect 83, an arrangement ofchannels and valves in a microfluidic device, comprising: (a) an inputmultiplexer that comprises: (i) a plurality of four or more inputchannels; (ii) a plurality of input valves configured and arranged tocontrol the input channels such that fluid in any one of the inputchannels may flow independently of fluid in the other input channels;(iii) one or more combining channels configured and arranged to receivefluid flowing through any one or more of the input channels and to sendthe fluid through a single connecting channel; (b) the connectingchannel; and (c) an output multiplexer that comprises: (i) a pluralityof four or more output channels; (ii) a plurality of output valvesconfigured and arranged to control the output channels such that fluidin any one of the output channels may flow independently of fluid in theother output channels; (iii) one or more distributing channelsconfigured and arranged to receive fluid flowing through the connectingchannel and to send the fluid through any one or more of the outputchannels depending on operation of the output valves.

In one aspect, the application discloses Aspect 84, the arrangement ofaspect 83, wherein the one or more combining channels is a manifold.

In one aspect, the application discloses Aspect 85, the arrangement ofaspect 83 or 84, wherein the path length from any one of the inputchannels through the combining channels to the connecting channel is thesame.

In one aspect, the application discloses Aspect 86, the arrangement ofany of aspects 83-85, wherein the one or more distributing channels is amanifold.

In one aspect, the application discloses Aspect 87, the arrangement ofany of aspects 83-86, wherein the path length from the connectingchannel through the distributing channels to any one of the outputchannels is the same.

In one aspect, the application discloses Aspect 88, the arrangement ofany of aspects 83 to 87, configured so that a eukaryotic cell may passintact through and from any one of the input channels, through thecombining channels, through the connection channel, through thedistributing channels, and through any one of the output channels.

In one aspect, the application discloses Aspect 89, the arrangement ofany of aspects 83-88, wherein each of the input channels connects to andis configured to receive fluid from a different region of a partitioningchannel.

In one aspect, the application discloses Aspect 90, the arrangement ofaspect 89, wherein the input valves are positioned between thepartitioning channel and the combining channels.

In one aspect, the application discloses Aspect 91, the arrangement ofaspect 89, wherein the partitioning channel is positioned between theinput valves and the partitioning channel.

In one aspect, the application discloses Aspect 92, the arrangement ofany of aspects 83-91, wherein a plurality of the output channels eachconnects to a separate holding chamber.

In one aspect, the application discloses Aspect 93, the arrangement ofaspect 92, wherein the holding chamber is configured for cell culture.

In one aspect, the application discloses Aspect 94, the arrangement ofany of aspects 91-93, wherein the channels in the input manifold, theconnecting channel, and the channels in the output manifold are allfilled with fluid.

In one aspect, the application discloses Aspect 95, an apparatusconfigured to support and operate a microfluidic device, comprising: (a)a platform that comprises: (i) a cavity shaped and sized to receive andsupport a microfluidic device; (ii) optically transparent (e.g., glass)window positioned above the cavity so that when cells are beingprocessed by a device in the cavity, the cells may be imaged through thewindow; (iii) an interface plate that comprises a plurality of openingsconfigured to seal to control channels in a microfluidic device in thecavity and to operate valves in the device by pneumatic pressure; (iv)an integral heating member that surrounds the cavity in the plane of theplatform, configured to maintain a microfluidic device in the cavity ata temperature suitable for cell culture; (b) a mix box that comprises:(i) an inlet configured to receive a gas mixture; (ii) a humidifierconfigured to humidify the gas mixture; (iii) a heater configured toheat the gas mixture to a temperature suitable for cell culture; and (c)a conduit configured so that gas heated by the heater in the mix box maypass through the platform into a microfluidic device in the cavity.

In one aspect, the application discloses Aspect 96, the apparatus ofaspect 95, further comprising: (d) a conduit configured so that spentgas from a microfluidic device in the cavity may pass back to and outthrough the mix box; and (e) a conduit configured so that spent liquidfrom a microfluidic device in the cavity may pass back to and outthrough the mix box.

In one aspect, the application discloses Aspect 97, the apparatus ofaspect 95 or 96, wherein the platform further comprises: (v) a humiditysensor configured to determine humidity of a gas mixture passing intoand/or out of a microfluidic device in the cavity.

In one aspect, the application discloses Aspect 98, the apparatus ofaspects 95-97, wherein the mix box further comprises: (iv) a luer lockconnecter configured to connect the inlet to a source of gas mixture;and (v) an electrical socket.

In one aspect, the application discloses Aspect 99, the apparatus of anyof aspects 95-98, wherein the integral heating member passes from theplatform to the mix box so as to heat a microfluidic device in thecavity and a gas mixture in the mix box at the same time.

In one aspect, the application discloses Aspect 100, the apparatus ofany of aspects 95-99, wherein the integral heating member comprisespolyamide.

In one aspect, the application discloses Aspect 101, the apparatus ofany of aspects 95-100, wherein the optically transparent (e.g., glass)window is coated with a coating that inhibits condensation.

In one aspect, the application discloses Aspect 102, the apparatus ofaspect 101, wherein the coating comprises iridium and tin.

In one aspect, the application discloses Aspect 103, a microfluidicsystem comprising a support apparatus according to any of aspects 95-98with a microfluidic device in the cavity.

In one aspect, the application discloses Aspect 104, the microfluidicsystem of aspect 103, wherein the microfluidic device is a device ashereinbefore described.

In one aspect, the application discloses Aspect 105, the microfluidicsystem of aspect 103 or 104, further comprising a supply of a gasmixture and a supply of nutrient medium for culturing cells in themicrofluidic device.

In one aspect, the application discloses Aspect 106, the microfluidicsystem of any of aspects 103-105, further comprising an imagingapparatus configured to capture images of cells in the microfluidicdevice.

In one aspect, the application discloses Aspect 107, the microfluidicsystem of any of aspects 103-106, further comprising a computerprocessor configured and programmed to control operation of themicrofluidic device.

In one aspect, the application discloses Aspect 108, the microfluidicsystem of aspect 107, wherein the computer processor is a dedicatedprocessor.

In one aspect, the application discloses Aspect 109, the microfluidicsystem of aspect 107, wherein the computer processor is a processor in aportable computer system comprising an app coded to control operation ofthe microfluidic device.

In one aspect, the application discloses Aspect 110, the microfluidicsystem of aspects 107 to 109, wherein the computer processor isconfigured and programmed to display images of cells in the microfluidicdevice.

In one aspect, the application discloses Aspect 111, use of apartitioning channel in a microfluidic device as heretofore described inthe processing of a cell population.

In one aspect, the application discloses Aspect 112, use of an inputmultiplexer connected to an output multiplexer in a microfluidic deviceas heretofore described in the processing of a cell population.

In one aspect, the application discloses Aspect 113, use of a pluralityof holding chambers in a microfluidic device as heretofore described inthe processing of a cell population.

In one aspect, the application discloses Aspect 114, use of a supportapparatus for a microfluidic device as heretofore described in theprocessing of a cell population.

In one aspect, the application discloses Aspect 115, use according toany of aspects 111-114, wherein the processing comprises separating andsorting individual cells (or cellular units) from the cell population.

In one aspect, the application discloses Aspect 116, use according toany of aspects 111-115, wherein the processing comprises separatelyculturing individual cells (or cellular units) from the cell population.

In one aspect, the application discloses Aspect 117, use according toany of aspects 111-116, wherein the processing comprises separating andcombining individual cells (or cellular units) from the cell populationwith other individual cells (or cellular units) separated from the cellpopulation.

In one aspect, the application discloses Aspect 118, use according toany of aspects 111-117, wherein the processing comprises separatelycollecting contents of individual cells (or cellular units) from thepopulation and subjecting the contents of each cell to a chemicalreaction.

In one aspect, the application discloses Aspect 119, use according toaspect 118, wherein the chemical reaction is part of an assay.

In one aspect, the application discloses Aspect 120, use according toany of aspects 118-119, wherein the processing comprises separatelyimaging individual cells (or cellular units) from the population.

In one aspect, the application discloses Aspect 121, a method ofdetermining properties of a population of cells comprising providing anacellular composition comprising macromolecules from exactly N cells,where N=2, wherein said N cells have been cultured together for at least2 hours, and carrying out an analytical step on said macromolecules.

In one aspect, the application discloses Aspect 122, the method ofaspect 121 wherein the macromolecules are nucleic acids and theanalytical step comprises amplification, transcription, reversetranscription, cloning or sequencing.

In one aspect, the application discloses Aspect 123, the method ofaspect 121 wherein the macromolecules are proteins and the analyticalstep comprises combining the proteins with an antibody.

In one aspect, the application discloses Aspect 124, the method of anyof aspects 121-123 except N=3, 4, 5, 6, 7, 8 or is less than 10.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart showing exemplary steps for carrying out theinvention.

FIGS. 2A and 2B shows schematic of embodiment of the invention includinga partitioning channel(s) and valves spaced along a length of thechannel(s). The multiplexor is not shown. FIG. 2A shows an embodimentwith one partitioning channel and FIG. 2B shows an embodiment with twopartitioning channels.

FIGS. 3A and 3B illustrate the principal of multiplexors that may beused to independently transport a single cell from any segment to anypredetermined destination chamber. FIGS. 3A and 3B illustrate twopositional relationships of the partitioning channel and multiplexors.

FIG. 4 shows an exemplary design of a device. FIG. 4A shows a fluidicsystem in which channels of the combining manifold, and similarlychannels of the distributing manifold, are separated into two blocks,each block remaining connected via the secondary manifold systems toprovide a single unified combining manifold and, similarly, a singleunified distributing manifold connected by a connector channel. FIG. 4Bis a detail from FIG. 4A.

FIG. 5 illustrates a relationship, in one embodiment, of a segment, aflow channel and a multiplexor channel.

FIGS. 6A-6C show exemplary designs of destination chambers.

FIG. 7 shows an exemplary design of a device.

FIG. 8 shows a multi-chamber reaction configuration that may befluidically connected to the cell holding chamber.

FIG. 9 shows a virtual screen shot of the graphical user interface ofthe system.

FIG. 10 shows a microfluidic system including an interface plate toenable control of the internal portions the PDMS IFC (e.g., valves andchannels) as well as control the environmental conditions surroundingthe PDMS chip, e.g., humidity, temperature and gas composition.

FIG. 11 shows an illustrative diagram of a drain.

FIGS. 12 and 13 show an alternative path for transporting a cell from asegment to a cell holding chamber.

FIGS. 14 and 15 illustrate embodiment in which more than one connectorchannel is used.

DETAILED DESCRIPTION OF THE INVENTION 1. Introduction

The present invention relates to methods and devices for analysis ofindividual cells (or cellular units) and defined combinations of cells.The method includes using a microfluidic system for carrying out severalrounds of cell capturing, characterization, and transport. Cells andcombinations of cells are cultured together and may be subject tofurther analysis or manipulation including genotyping, nucleic acidamplification and preamplification, analysis of gene expression,treatment with miRNA, analysis (e.g., activity assays) of DNA andprotein targets.

In one aspect, the invention provides a method for cell analysis bycomprising carrying out at least two rounds of cell capturing,characterization, and transport, each round comprising a) flowing asolution comprising a plurality of individual cells (or cellular units)into a first microfluidic channel; b) partitioning the channel into aplurality of contiguous segments, thereby capturing at least one cell inat least one segment, wherein one or more of said segments comprises asingle captured cell or single captured cell unit; c) determining atleast one characteristic of one or more of said single captured cells orsingle captured cell units; and d) independently transporting each saidcaptured cell or single captured cell unit to a specified destinationchamber based on the determined characteristic, whereby for eachspecified destination chamber the characteristic(s) of cell(s)transported thereto is known. Cells and combinations of cells areanalyzed directly, cultured and, optionally, harvested for molecularbiological processing. See FIG. 1.

A variety of methods and devices for cell isolation and manipulation aredisclosed in U.S. patent application Ser. No. 13/781,292, filed Feb. 28,2013 and published as US 2013-0302883 A1, which is incorporated hereinin its entirety all purposes.

2. Definitions

“Plurality” means at least three.

“Majority” means more than 50%.

A “cell” may be a eukaryotic cell or a prokaryotic cell.

An “individual cell” is a cell that is not in physical contact withother cells, e.g., is not part of a solid tissue or multicellularstructure. An individual cell may be one that in its naturally state is,at least for a portion of its life, not in contact with other cells(e.g., circulating lymphocytes; spermatozoa, oocytes, nucleated redblood cells, single cell algae, protozoa and the like). Alternatively,an individual cell may be one that in its naturally state is generallyin contact with other cells (e.g., hepatocytes, neurons) but which hasbeen separated from those other cells (e.g., by disaggregation of atissue).

An “individual cellular unit” is an aggregation of a small number ofcells (e.g., 2, 3, 4, 5 cells, or less than 10 cells and/or aggregationof a small number of cells having a size capable of flowing through amicrofluidic channel with a 1000 micron diameter lumen). Forillustration, a pluripotent stem cell aggregate may be a cellular unit.

The phrase “individual cells (or cellular units)” should be read as “Insome embodiments, individual cells; In other embodiments individualcellular units” and “In still other embodiments a mixture of individualcells and cellular units. Likewise, the phrase “single captured cells orsingle captured cell units” should be read as “In some embodiments,single captured cells; In other embodiments single captured individualcellular units” and “In still other embodiments a mixture of singlecaptured cells and single captured cellular units.”

Two components of a microfluidic device are referred to as “fluidicallyconnected” when they are connected by a microfluidic channel or otherconduit that can carry fluid from one of the components to the other.

“Valves” are used to restrict movement of cells and/or movement of fluid(air or liquid) through a channel. As discussed herein below (§ 17A),valves may have a variety of designs and structures. In addition, valvesmay be referred to by function (e.g., “partition valves,” “upstreamvalves,” downstream valves,” multiplexor valves,” “permeable valves,”“one way valves,” and “pump valves”).

A “channel filter” refers to a non-actuatable barrier in a channel(e.g., an upstream channel or a drain channel) that prevents passage ofcells through or into the channel but allows liquid to flow through. Insome embodiments the filter is made by packing a portion of the channelwith beads or comprises frits, posts, porous polymer monolithicmaterials, and the like.

A “length” of a microfluidic channel refers to a channel or portion of achannel into which cells are introduced and which is partitioned into aplurality of contiguous segments.

“Capturing” refers to using a physical barrier to confine a cell to aparticular segment of a channel

As used herein, “address” refers to a location in a microfluidic system.For example, in a microfluidic system of the invention each segment maybe assigned an address (e.g., 1-10) and each cell holding chamber may beassigned an address (e.g., a-z).

3. Partitioning Channel

Individual cells (or cellular units) may be isolated by flowing acomposition comprising a plurality of cells into a microfluidic channel(referred to as a “partitioning channel”) and physically isolating atleast one individual cell from other cells in the channel. The step ofphysically isolating may be accomplished by partitioning thepartitioning channel into a plurality of segments, at least one of whichcontains a single cell. FIG. 2A illustrates a partitioning channel andvalves spaced along a length of the channel. When the valves areactuated, the channel is partitioned into a plurality of segments. (InFIG. 2A and certain other schematic figures, the positions of theselected values are illustrated using elongated solid rectangles. Itwill be apparent from context which valves are open or closed during aparticular operation. Not all valves are shown.)

The dimensions of the partitioning channel may vary depending on thechannel materials, valve design, and other factors, but in any eventdimensions are sufficient to allow a cell, generally a eukaryotic cell,to flow through the channel. Dimensions of eukaryotic cells aregenerally in the range of about 4 to about 100 microns. Exemplarycross-sectional dimensions for partitioning channels are about 15microns to about 1000 microns, about 15 microns to about 500 microns,sometimes about 500 microns to about 1000 microns, sometimes about 200microns to about 800 microns.

It will be understood and clear from context that reference to“partitioning channel” often refers to the portion of the partitioningchannel along which valves are actuated to make segments. It will beunderstood that the partitioning channel will usually have segments thatlack partition valves, typically at both ends, through which cells areintroduced or withdrawn from the channel. In some embodiments, themicrofluidic system comprises multiple (e.g., two) partitioningchannels. See § 4, below.

4. Introducing Cells into the Partitioning Channel

In a first step in the process of the invention, a solution containingcells is flowed into the partitioning channel, and into the length ofthe channel in which cells are captured. Generally the cells are in asolution in which the cells are viable, such as a cell culture medium.It is within the ability of a person of ordinary skill in the art toidentify an appropriate medium for cells being studied. In someembodiments the cells are maintained at a specified temperature (e.g.,room temperature or 37 degrees C.), humidity, and/or atmosphere (e.g.,CO₂/O₂ partial pressure).

Prior to introducing cells into the partitioning channel they may bestored in a reservoir that is permanently or reversibly in fluidcommunication with the partitioning channel.

Cells are generally introduced into the partitioning channel by bulkfluid flow, as described in § 9 below but may be introduced into thepartitioning channel using other methods of transport, as known in theart and also described in in § 9 below.

In some embodiments, the microfluidic system comprises multiple (e.g.,2) partitioning channels. See FIG. 2B. A device with multiplepartitioning channels may have advantages when two distinctly differentcell populations or types are being analyzed and combined, especiallycells that are of significantly different sizes, cells for whichdifferent characteristics are being determined, cells that requiredifferent media, experiments in which cross-contamination between cellpreparations must be avoided, and the like.

Alternatively, as discussed below, different cells populations or typescan be introduced into the same partitioning channel at different times(e.g. sequentially). Alternatively, different cell types, or differentcell populations can be mixed and selected after capture based ondetermined characteristics. Frequently, a sample is obtained thatcontains a mixture of different cell types and the methods and systemsof the invention are used to select individual cells (or cellular units)of interest from the mixture, usually discarding the other cells.

In some embodiments the cells of interest are rare in the population.For example, the in the solution introduced into the partitioningchamber the ratio of cells of interest to other cells may be greaterthan 100:1, sometimes greater than 1000:1, sometimes greater than10,000:1, and sometimes greater than 100,000:1. Examples of rare cellsinclude stem cells, tumor cells (e.g., a circulating tumor cell),circulating endothelial cells, and fetal cells.

5. Valves, Partitioning, Segments

As noted above, valves are spaced along a length of the partitioningchannel, which valves may be actuated to partition the channel into aplurality of segments.

As used herein, the term “valves” refers to structures used to restrictmovement of cells and/or movement of liquid through a channel. Valvesare actuatable (can be closed or opened in response to an actuationsignal) and generally reversible (can both be closed and opened inresponse to a signal or signals). As discussed herein below in § 17A,valves may have a variety of designs and structures. In addition, valvesmay be referred to by function (e.g., “partition valves,” “upstreamvalves,” downstream valves,” multiplexor valves,” “permeable valves,”“one way valves,” and “pump valves”).

Some valves stop movement of liquid through a channel (e.g., bycompletely blocking the channel lumen). These valves will also blockmovement of cells though the channel. “Permeable” valves block cellmovement but allow liquid to flow through them. For example, a partiallyclosed valve can block enough of the lumen to prevent cell passage whileletting fluid through. One approach to partially closed valves is foundin, e.g., US Pat. Pub. 20080264863 to Quake (describing sieve valves).Another permeable valve is a valve having pillars integral to themembrane structure, so that when the valves are closed the pillarsmaintain gaps that allow fluid, but not cells, to flow through. See,e.g., FIG. 27AB of U.S. Pat. No. 7,291,512. Nonactuatable channel“filters” are described below, and also allow block cell movement butallow liquid to flow through.

Valves that partition the partitioning channel may be called “partitionvalves.”

The partitioning of the partitioning channel into segments is themechanism by which individual cells (or cellular units) are physicallyseparated from other cells. Two cells are physically separated whenthere is a physical barrier interposed between them and the cell areeach confined to a unique space. Typically cell capturing is astochastic process based on random distribution of cells in thepartitioning channel.

The partitioning channel may be substantially featureless, or may havesecondary features used to position cells to increase the likelihoodthat segments will contain individual cells (or cellular units). Anexample of a secondary feature is found in FIG. 17 and Example 5 of USPat. Pub. 20100120077 describing a “cell comb” that could be used toposition individual cells (or cellular units) in a channel (e.g.,channel 616) prior to partition. Another example is a channel withsignificant and periodic differences in width along its length (that, byaffecting flow speed, may increase the deposition of particles in theslower regions.

In contrast a substantially featureless channel has generally a constantlumen dimension and shape. Although embodiments with secondary featuresare contemplated, a substantially featureless partitioning channel ispreferred.

As noted, when the valves are actuated, the channel is partitioned intoa plurality of segments. Usually the segments are contiguous. In oneembodiment the valves are spaced substantially equally along the lengthof the channel, resulting in segments that are about equally sized.

The number of segments generated can vary across a broad range, e.g.,from 10-200, often from 50-100, and sometimes more than 100. Generallythere are at least 25 segments, sometimes at least 50 segments, andsometimes at least 75 segments.

The size (volume and dimensions) of the segments can vary over a largerange. The segments should be large enough to contain an individual cellbut small enough for efficient use of space. Exemplary segment lengthsare about 20, about 30, or about 40 microns in length (e.g., about 20 toabout 40 microns in length).

In some embodiments, the volume of each segment is many times that of asingle target cell. The volume of most eukaryotic cells is in the rangeof 0.05 picoliter to 4.2 nanoliters.

In another embodiment, the fluid flow does not stop and various cellsand a variety of positions are sorted (i.e. parallel shift register).This process requires tight coordination of valve actuation, but has theadvantage of faster cell selection of multiple cells in a parallel mode.

6. Cell Concentration

The invention may be used to characterize and combine single cells,single cell units, or combinations. It is desirable therefore that atleast some partitioned segments contain a single cell and it isgenerally advantageous to maximize, to the extent practical, theproportion of partitioned segments that contain a single cell (incontrast to segments that contain no cell or contain multiple cells).The size of segments, concentration of cells, and other factors may beselected to selected to maximize capturing of single cells. An optimalcell concentration for introduction into the partitioning channel, forexample, can be determined empirically or can be calculatedmathematically.

In determining that a segment contains a single cell, in someembodiments, when the presence of other cells does not interfere withthe analysis to be conducted, cells of a particular class are counted.For example, if a solution containing a few eukaryotic cells and a vastexcess of bacterial cells is used, some segments will contain a singleeukaryotic cell, and some or all segments would likely contain manybacterial cells. In this case a segment containing one eukaryotic celland numerous bacterial cells may be considered to have a single cell (ofthe specified class). Similarly, if a solution containing a fewnucleated cells and a vast excess of anucleate red blood cells is used,some segments will contain a single nucleated cell, and some or allsegments would likely contain many erythrocytes. In some cases, the cellpopulation of interest are (i) all eukaryotic cells, (ii) all nucleatedcells, (iii) all animal cells, (iv) all human cells, (v) all prokaryoticcells, (vi) all protozoa, (vii) all parasites, (viii) all fungal cells.

As discussed below, it is contemplated that multiple rounds of cellcapturing will be carried out to populate a plurality of cell holdingchambers with cells of interest. Preferably, each round of cellcapturing results in at least one segment with a single cell. Usuallythere will be at least one segment with no cell. Preferably at least30%, alternatively at least 40%, and preferably the majority of segmentscontain no more than one cell (i.e., they contain one cell or zerocells).

7. Determining at Least One Characteristic of a Captured Cell

One or more characteristics or, equivalently, “properties,” of thecaptured cells (i.e., some or all of the captured cells) are determinedwhile the cells are captured in the segment. Examples of characteristicsthat may be determined include, for example and not limitation, size,morphology, optical properties (e.g., color, refractive index; see,e.g., Coelho et al., 2006, “Measuring optical and mechanical propertiesof a living cell with defocusing microscopy,” Biophys J. 91:1108-15),presence or absence of an extracellular or intracellular antigen,nucleic acid content, cell membrane electrical properties, mobility,response to stimulus, etc.

The method of determining the characteristic will depend on the specificcharacteristic(s) detected. For example, cell properties can bedetermined optically (e.g., using a microscope or CCD camera),spectroscopically (e.g., using Raman spectroscopy), by measuringdielectrophoretic properties (see, e.g., K. Hoettges 2008,“Dielectrophoresis as a Cell Characterisation Tool” in Methods inMolecular Biology 583:183-198), or by detecting a signal (e.g.,fluorescent signal) associated with a cell antigen or nucleic acid.Devices for detecting the cell property can be wholly or partly integralto the microfluidic device or may be external to the device. Thedetermination process can manual, or partly or wholly automatic.Exemplary signal detectors monitor visible, fluorescent, and UV light(intensity, scattering, absorption) luminescence, differentialreflectivity, electrical resistance, resistivity, impedance, andvoltage. Applications can also utilize confocal laser scanning,radiochemical detection, fluorescence polarization and other methods. Itwill be recognized that the design and materials used in construction ofthe microfluidic system will be compatible with the mechanism(s) ofdetection. For example, when optical methods are used, the partitioningchannel may be manufactured using materials transparent to at least thewavelengths of light required for detection. Alternatively, fiber opticand other systems may be employed.

Captured cells may be characterized by a combination of more than onecharacteristic, such as a combination of the presence or absence of aseveral extracellular antigens, or intracellular biomarkers includingantigens (proteins), specific RNA or DNA sequences, or protein presenceor activity.

Different populations of cells of interest may be identified by assayingcaptured cells for two different characteristics, one a characteristicof one population and the second a characteristic of a secondpopulation. In one approach at least one detectable label is associatedwith cells in a population based on a property present in (positiveselection), or absent from (negative selection), cells of interest.Suitable immunocytochemical methods for characterizing cells are verywell known in the art.

For illustration and not limitation, cell characteristics include threecategories of properties, any of which may be used to characterizecells. (1) characteristics of untreated cells; (2) detectable labelsassociated with cell components; and (3) cell responses to challenges.

Characteristics of untreated cells can include morphology, opticalproperties, electrical properties, metabolic properties (e.g., O₂consumption), behavior (mobility, membrane ruffling, and the like).

Detectable labels associated with cell components can include detectablelabels associated with nucleic acids, cell antigens, and other cellcomponents. A noncomprehensive list of exemplary detectable labels isprovided in § 17(B).

Cell responses to challenges refers to detection of a cell property thatchanges in response to a change in the physical or chemical environmentof the cell. For example, captured cells may be exposed to a physicalchange (temperature change, illumination, pH change, etc.) and theresponse of the cell detected. As another example, captured cells may bechallenged by exposure to a reagent (e.g., drug, test agent, inhibitor,etc.) and the cell response detected.

It will be appreciated that characteristics of untreated cells and cellresponses to challenges may be detected using a detectable label. Forexample, the endocytosis (as a cell property) or increased endocytosisin response to a physical or chemical challenge, can be detected byexposing the cells to incubating the cells with fluorescently labeledpolystyrene nanoparticles and detecting update of the fluorescent label.

Cells can be challenged and/or labeled with a detectable label prior tointroducing cells into the partitioning channel, after the cells areintroduced into the partitioning channel, or both.

For example, in some embodiments at least some cells are detectablylabeled prior to introduction into the partitioning channel. In otherembodiments, at least some cells are labeled while captured in thechannel, e.g., by flowing a detection reagent through the channel. Acell population may be labeled with one label before capturing andlabeled with a different label after capturing. A cell population may belabeled by exposing the cell to one reagent before capturing and to acorresponding detectable label after capturing. For example, a cellpopulation may be exposed prior to capturing to a mouse antibody thatrecognizes a cell surface antigen and then exposed while captured to arhodamine-tagged anti-mouse antibody to make the cell.

Single captured cells determined to have a property of interest aretransported to cell holding chambers. The remaining cells may bediscarded, for example, by opening the valves in the partitioningchannel and removing the cells and other material not transported tocell holding chambers. In one embodiment, several queries can be made toselect cells for transport to cell destination chambers. For example andnot limitation, the following quires can be made for each segment:

8. Multiplexor Channels and Flow Channels Associated with Segments

Captured cells identified as having appropriate characteristics aretransported to one of several cell destination chambers (described in §10 below). Any transport system that permits selected single cells (orsingle cell units) to be independently transported to specified celldestination chambers may be used. As used in this context,“independently transported” means that an individual cell may betransported from any one of a plurality (e.g., 10-500) of segments toany one of a plurality (e.g., 10-500) of cell holding chambers, allowingselected combinations of cells to be transported to the same celldestination chamber.

In one approach, cells are transported using a compound manifold system.The compound manifold comprises a “combining manifold” and a“distributing manifold” connected by a connector channel X. Each of thesegments S of the partitioning channel is in fluid communication withunique combining channel Cc of the combining manifold. Flow through eachunique channel Cc can be controlled by actuating selected valves orotherwise controlling flow (or cell movement in the case ofelectro-osmotic transport) through each channel Cc, so that cells canflow through only a single selected channel Cc. Each channel Cc isfluidically linked to a connector channel X. The combining channels Ccmay be linked to connector channel X by a single common channel (asillustrated schematically in FIGS. 2 and 3), by a secondary manifoldsystem (as illustrated in FIGS. 4A, 4B, and 7), by more than oneconnector, or otherwise. (The terms “combining manifold” or“distributing manifold” encompass the associated secondary manifoldsystems.) The system is arranged so that when channels are configured toallow particles (e.g., a cell) to flow through only one combiningchannel Cc, the particles will flow into the connector channel X.

The distributing manifold mirrors, to a degree, the combining manifold.Each of the cell holding chambers H is in fluid communication withunique distributing channel Cd of the distributing manifold. Flowthrough each unique channel Cd can be controlled by actuating selectedvalves or otherwise controlling flow (or cell movement in the case ofelectro-osmotic transport) through each channel Cd, so that cells canflow through only a single selected channel Cd. Each channel Cd isfluidically linked to connector channel X. The combining channels Cd maybe linked to connector channel X by a single common channel (asillustrated schematically in FIGS. 2 and 3), by a secondary manifoldsystem (as illustrated in FIGS. 4 and 7), or otherwise. The system isarranged so that when distributing channels are configured to allowparticles (e.g., a cell) to flow through only one distributing channelCd, the particles will flow from connector channel X to only one cellholding chamber H.

It will be appreciated by reference to the figures and descriptionherein that using a compound manifold, a cell can be independentlytransported from any segment S (i.e., any segment address in thepartitioning channel) to any to any cell holding chamber H (i.e., anycell holding segment address.

The “combining manifold” and associated valves, and the “distributingmanifold” and associated valves, each can be referred to as a“multiplexor” system.

In one approach, cells are transported using such a compound manifold ormultiplexor system. Single multiplexor systems are described in, forexample, U.S. Pat. No. 7,143,785, incorporated herein by reference. Alsosee, T. Thorsen “Microfluidic Technologies for High-Throughput ScreenApplications,” Ph.D. Thesis, California Institute of Technology, Chapt.5; and Melin and Quake, 2007, “Microfluidic Large-Scale Integration: TheEvolution of Design Rules for Biological,” Automation Annu. Rev.Biophys. Biomol. Struct. 36:213-31; Hua et al., 2006, A versatilemicroreactor platform featuring a chemical-resistant microvalve arrayfor addressable multiplex syntheses and assays. J Micromech Microeng16:1433-1443; each incorporated herein by reference. Microfluidicmultiplexors can switch flow from a plurality of individuallyaddressable inputs (channels) to a single output, or switch flow from asingle input to a plurality of individually addressable outputs.

Optionally the multiplexor system includes a shift register. (See Sai etal., 2102, “Pressure driven digital logic in PDMS based microfluidicdevices fabricated by multilayer soft lithography” Lab on a Chip 12,4809-4815; DOI: 10.1039/c2|c21155f). Optionally, the multiplexor systemoperates by control signals generated by a shift register wherein theshift register is controlled by an user-settable input, a repeating‘clock’, and a trigger valve. The user-settable input provides serialinput instructions that are ‘read’ into the shift register, moving theinput signal one ‘bit’ through the register per ‘clock’ cycle. Thetrigger valve serves to open communication between the pneumatic shiftregister outputs and the fluidic multiplexor ‘control’ channels.

FIGS. 3A and 3B illustrate the principal of a multiplexor system of theinvention. In these figures, elastomeric valves actuated by controlchannels 500 are represented. See Unger et at., 2000, “Monolithicmicrofabricated valves and pumps by multilayer soft lithography” Science288: 113-116. In the cartoon the control channels have wider regions andnarrower regions. Valves 600 are created where a wide section of acontrol channel crosses over (or under) a fluidic channel. By actuatingselected combinations of control channels paths may be created from anysegment to any cell holding chamber. It will be appreciated that thevalve pattern in FIGS. 3A and 3B are for illustration and that a varietyof other patterns could be used. See, e.g., FIG. 7. The elastomericsystem is highly efficient and allows control flow through n fluidchannels using <n control channels. However, it will be appreciated thatthe method is not limited to elastomeric embodiments. Virtually anynumber of valves that substantially completely stop fluid flow could beused. For example, in one approach flow through each multiplexor channelcan be controlled by an independently actuatable valves, and flow can becontrolled by, for example, closing all but one valve in the partitionchamber sector and all but one valve in the holding chamber sector.

In one embodiment, bulk fluid flow is used to transport selected cellsfrom a segment to a cell holding chamber. For illustration, withreference to see FIG. 5, each partitioning channel segment is in fluidcommunication with a multiplexor channel 107 and a flow channel 105. Themultiplexor channel may be a downstream multiplexor channel (FIG. 3A,FIG. 7) or an upstream multiplexor channel (FIG. 3B). The flow channel105 may be an upstream flow channel (FIG. 3A, FIG. 7) or a downstreamflow channel (FIG. 3B). Downstream channels lie between the partitioningchannel and the cell destination chambers. In each approach, thedownstream channel has appropriate dimensions and is otherwise suitablefor carrying cells (i.e., cells flow through the downstream channel). Incontrast, the upstream channel provides liquid that facilitates flow ofthe cell into the downstream channel. It will be recognized that adownstream multiplexor channel is used with an upstream flow channel andvice versa.

Following partition, a cell may be transported by a fluid streamfollowing the path: (1) fluid source→(2) upstream channel→(3)segment→(4) downstream channel→(5) connector (e.g., connectionmanifold)→(6) holding chamber sector flow channel→(7) cell holdingchamber→(8) (optionally) multi-chamber reaction configuration.

As illustrated in FIG. 5, in addition to valves used to effect thelongitudinal separation resulting from partition of the partitioningchannel, additional valves may be optionally used to control flow ofcells into upstream channels (upstream valves 106) and/or downstreamchannels (downstream valves 104). For example, cells may be flowedthrough the partitioning channel with partition valves 102 open, andvalves 104 and 106 may be closed to prevent the cells from flowing intoupstream and downstream channels. Alternatively upstream and/ordownstream channels may not have valves. For example, upstream channelsmay have channel dimensions or geometry that prevent a cell fromentering the channel. In another approach, they may have channel filterswhich allow liquid to flow through them but block passage of cells. Inanother approach, in the case of an upstream multiplexor, themultiplexor valves when closed will block the upstream channel. In thiscase, even if cells migrate into the upstream channels they can betransported out of those channels, either to a destination chamber or towaste. It will be recognized that this design may make the step ofremoval of unselected cells more complex. In another approach, channeldimensions can be selected to render upstream and/or downstream valvesunnecessary. This is because, during the loading step in which cells areflowed into the partitioning channel, there will be flow though thepartitioning channel but no flow (“static flow”) through the rest of thesystem; as a result cells flowing through the partitioning channel tendnot to drop into upstream or downstream channels, which are occupied byfluid.

Generally each segment is in communication with a different (unique)multiplexor channel. Alternatively, it is possible to use manifoldsconnecting pairs of segments so that pairs or multiples of channels areconnected to a distributing multiplexor channels (used, for example,when only one segment of a connected pair comprise a target cell)although such designs generally require a sacrifice in efficiency.

FIG. 7 illustrates an exemplary design of the multiplexor system. Thesystem comprises partitioning channel 100, partitioning valves 110,upstream flow channels 105, partitioning channel selector (multiplexor)200 comprising downstream multiplexor channel 101 and downstream valves104, secondary manifold systems 300, connector 400 (not shown), cellholding chamber channel selector (multiplexor) 500, and cell holdingchambers 600. Also shown are control channels 700 controllingcontainment valves.

In contrast to the schematic multiplexor shown in in FIG. 3, secondarymanifold systems are shown in FIG. 7. The manifolds ensure that thedistance (and approximate transit time) between any segment and any cellholding chamber is the same. This uniformity has the advantage ofmaintaining a consistent actuation time and routines between valvedpartitions that are transporting a cell from any segment to any cellholding chamber.

FIGS. 14 and 15 illustrate embodiment in which more than one connectorchannel is used. In FIG. 14 each of two combining manifolds is connectedby a separate connector (C₁ and C₂) to a single distribution manifold(compare FIG. 2B). FIG. 15 illustrates a system with two connectors, C₁and C₂. At time N, a cell is transported through C₁ and enters a cellholding chamber. At time N+1, a different cells is transported throughC₂ and enters a different cell holding chamber.

Another approach for transporting cells from a segment to a cell holdingchamber is illustrated in FIG. 12. In this approach cells are introducedinto a partitioning channel 100 and confined to segments 102 bypartitioning valves 101, as described above. The content of a selectedsegment (i.e., a selected single cell or group of cells). Selected cellsare transported through a combining manifold 200 to connector 300,generally as described above. The approach illustrated in FIG. 12differs from that of, e.g., FIG. 7 in the transport of cells from theconnector to a specified cell holding chamber through the distributingmanifold 500. As shown in FIG. 12, the connector channel is connected toa “second partitioning channel.” To avoid confusion, this secondpartitioning channel is referred to as the “stop channel” 400 but thefeatures of the stop channel are analogous to those of partitioningchannels described above. The stop channel comprises independentlycontrollable stop valves 401 and CHC valves 402. FIG. 13 illustrateshow, by actuating a selected stop valve and selected CHC valves, flow isdirected to a specific cell holding chamber (arrow). In FIGS. 12 and 13one stop valve is thickened to illustrate that it is closed such that acell is directed towards a particular chamber.

9. Flowing

Fluid and cells may be transported into and though channels in a varietyof ways known in the art. In general, transport of cells in the presentinvention occurs by bulk flow (i.e., the cells are carried in a streamof moving fluid). Flow of solution can be accomplished by a variety ofmethods. For illustration, the solution may be introduced by pumping(e.g., using a peristaltic pump as described in U.S. Pat. No. 6,408,878,incorporated herein by reference), by generating a positive pressuredifferential in the channel (e.g., cells may be introduced into thepartitioning channel from a syringe by depressing the plunger therebycreating a pressure differential) or a negative pressure differential(e.g., a syringe may be used to draw out fluid from a channel, causingflow through the channel), by gravity driven flow, or any other method.

In alternative embodiments cells are moved using electrophoretic,electrokinetic, or electro-osmotic methods. (see, e.g., Glawdel and Ren,2009, “Electro-osmotic flow control for living cell analysis inmicrofluidic PDMS chips” Mechanics Research Communications 36: 75-81,incorporated herein by reference).

10. Cell Holding Chambers and Cell Culture

10.1. Cell Holding Chambers

Systems of the invention have cell holding chambers for culture ofindividual cells (or cellular units) and combinations of cells captured,characterized and transported as described hereinabove. The number ofcell holding chamber may vary over a wide range, but is typically in therange of 10-500, more often 100-500, more often 150-400, and often about300 microns. The number of cell holding chambers may be smaller than,equal to, or greater than the number of segments. Generally the numberof cell holding chambers is smaller.

Minimum attributes of microfluidic cell holding chambers are two-fold:The chamber has to be large enough to contain at least one cell, andpreferably at least 2-10 cells, and the chamber has to be compatiblewith cell viability. The requirements for viability will vary from cellto cell (e.g., a human tumor cell will differ from a human hepatocyte oran algal cell), but typically the chamber must be maintained at anappropriate temperature and humidity, nutrients must be provided thecell(s), and waste products must be removed to the extent required forviability. The chambers typically include an inlet and an outlet toallow fluid flow in and out of the chambers. Nutrients that support cellgrowth can be either supplied from the inlet or the outlet channel.

Some or all of the surfaces of a culture chamber, such as the walls,roof, and/or substrate, may be treated or modified to facilitate aspectsof cell culture, particularly specific or nonspecific cell attachment,cell survival, cell growth, and/or cell differentiation (or lackthereof), among others. The cell holding chamber surface or base can bemodified to include a surface that supports cell growth, mimicking theextracellular matrix (ECM) of a tissue. Examples of typical ECMsinclude, but are not limited to, fibronectin, collagen, elastin,laminin, hyaluronic acid, heparan sulfate, chondroitin sulfate, keratansulfate.

The cell culture mechanism may culture cells under any suitableenvironmental conditions using any appropriate environmental controlmechanisms. Suitable environmental conditions may include a desired gascomposition, temperature, rate and frequency of media exchange, and/orthe like. Environmental control mechanisms may operate internal and/orexternal to a microfluidic system. Internal mechanisms may includeon-board heaters, gas conduits, and/or media reservoirs. Externalmechanisms may include an atmosphere- and/or temperature-controlledincubator/heat source, and/or a media source external to the system. Anatmosphere-controlled incubator may be more suitable when the system isat least partially formed of a gas-permeable material, such as PDMS.

Culture chambers may have any shape or composition consistent with therequirements above.

10.2. Exemplary Cell Holding Chambers

FIGS. 6A, 6B and 6C show exemplary arrangements for a cell holdingchamber of this invention. The chamber is configured so that fluidcontaining cells flow through an input channel, and one or more drainchannels act as an outlet that operates selectively to allow fluid todrain but not cells. The holding chambers depicted here aresubstantially round or circular in shape, with a plurality of drainchannels positioned around more than 180 degrees of the holding chamber,connected to a common output.

In some embodiments, the holding chambers of this invention may be 50microns to 2 mm in diameter, or between 100 and 500 microns in diameter.The dimensions are chosen according to the size and number of cells theoperator wishes to process in each chamber. For culturing or maintenanceof live cells, the holding chambers have a gas permeable membrane(typically above or below the chamber) so that the partial pressure ofgasses in the fluid may be maintained and adjusted appropriately. Theholding chambers may also be provided with a surface that promotes celladherence, such as an extracellular matrix (ECM) of proteins such asfibronectin and/or collagen, or a protein mixture produced from a cellline.

FIG. 6B shows a suitable arrangement of input and drain channels(showing a holding chamber that is 300 microns in diameter). FIG. 6Cshows an arrangement where the drain channels have been packed withbeads (i.e., “channel filters”) to further inhibit cells from flowingfrom the holding compartment to the drain.

FIG. 6A shows an arrangement where the input channel from themultiplexer tapers towards the holding chamber to form a focusingchannel. This configuration focuses each cell being delivered to theholding chamber, such that the cell tends to flow or migrate towards thecenter of the chamber. Distribution of a plurality of drain channelsabout the periphery of the chamber also helps maintain the cells at ornear the center of the chamber. This configuration tends to keep cellsat or near the center of the holding chamber, since draining is diffuseand the cells are not pulled towards the drain. In addition the abilityto confine the cells to these chambers is enabled by a combination ofelements including flow restrictors at the inlet (A), and/or at theoutlet (A and B). In addition the use of porous-to-liquid material, atthe outlet drains can be employed to prevent cells from exiting thechamber (C) The ability to control the fluid volume, pressure and sheerare important considerations to enabling cell viability. A third meansfor urging cells towards the center of the chamber is a concave shape inthe lower or supporting surface of the chamber upon which the cells arecultured. Positioning the cells at or near the center of the chamberhelps cells interact.

Cell culture media may be introduced into cell holding chambers throughthe same path taken by cells or through other channel. For example, withreference to FIG. 6, media may be introduced via a main input.Alternatively, media and other solutions may be introduced through otherroutes, such as via a branch channel that joins the input channel.

Drain channels are preferably openings low on the “wall” on the bottomsurface (“floor) of the cell holding chamber 100, but it is contemplatedthat they could be positioned elsewhere (e.g., on the floor of thechamber). As shown in FIG. 6 and FIG. 11, the drain 101 connects to themain drain 102. Preferably the length of the drain is short (e.g. about7.5 to about 25 microns). The width may be in the range, for example, ofabout 10 to about 25 microns, and the height of the drain channel ispreferably low (generally less than about 5 microns, sometimes less thanabout 4 microns, such as about 3 microns). To main drain to which thedrain channel connects is designed to have low fluidic resistance andmay have dimensions of 10-60 microns×20-40 microns. One design for adrain channel is shown in FIG. 11.

10.3 Introduction of Agents into Cell Holding Chambers

The system of the invention provides a powerful way to study the effectsof cell interactions (or co-culture) on metabolism, protein expression,and the like. In addition to media and nutrients, it will sometimes beuseful to add other agents to the cell holding chamber, to assess theresponse of the cell or cell combination to the agent(s) and/or toprovide reagents for assay of cell activity and the like. It will beappreciated that a variety of agents may be used, for example and notlimitation, chemical modulators or biological modulators, drugs,potential drugs (test agents), pathogens, proteins, antibodies, nucleicacids (e.g., DNA, RNA, modified nucleic acids, sense/antisenseexpression vectors, reporter genes, vectors for genomicintegration/modification antisense oligonucleotides, dsRNA, siRNA).Reagents for detection/assay reagents include, for illustration and notlimitation, dyes, enzymes, substrates, cofactors, ligands, antiligands,transfection reagents (e.g., lipid reagents, calcium phosphate, DMSO),polyethylene glycol, viral coats that package the nucleic acids, and/orso on.

10.4. Environmental Control

The environment of cell holding chambers is appropriate for theviability of the cell type(s) being cultured, including appropriatetemperature, humidity, pH, and gas composition.

10.5. Cell Culture

Microfluidic technology for culturing cells has been describedelsewhere. See Gomez-Sjöberg et al., 2007, “Versatile, fully automated,microfluidic cell culture system” Anal Chem. 79:8557-63; Zhong et al.,2008, “A microfluidic processor for gene expression profiling of singlehuman embryonic stem cells,” Lab Chip. 8:68-74; Glotzbach et al., 2011,“An information theoretic, microfluidic-based single cell analysispermits identification of subpopulations among putatively homogeneousstem cells,” PLoS One 6:e21211; Sanchez-Freire et al., 2012,“Microfluidic single-cell real-time PCR for comparative analysis of geneexpression patterns,” Nat Protoc. 7:829-38; U.S. Pat. No. 7,378,280“Apparatus and methods for conducting assays and high throughputscreening;” US 2010/0255471 “Single cell gene expression for diagnosis,prognosis and identification of drug targets.” The aforelistedpublications are hereby incorporated herein by reference in theirentirety for all purposes.

11. Rounds

It is contemplated that practice of the invention for analysis ofindividual cells (or cellular units) and combinations of cells willusually involve multiple rounds of flowing a solution comprising aplurality of individual cells (or cellular units) into a length of afirst microfluidic channel; partitioning the length into a plurality ofcontiguous segments thereby capturing at least one cell in at least onesegment; determining at least one characteristic of one or more of thesingle captured cells; and independently transporting captured cells tocell holding chambers. The number of rounds of capture will vary over awide range and will depend in part in whether the cells of interest arerare in the population of cells. Generally the number of rounds is inthe range of 2-1000. In exemplary embodiments the method comprises atleast two, at least three, at least 4, at least 5, at least 6, at least7, at least 8, or at least 9 rounds. In exemplary embodiments the methodcomprises 2 to 10 rounds, 3 to 10 rounds, 4 to 10 rounds, or 5 to 10rounds. In one embodiment the method comprises more than 50, more than100 or more than 200 rounds of capture and selection.

12. Discarding Cells

Between rounds, unselected cells remaining in the partitioning channelafter transport of selected cells to cell holding chambers can beremoved. One approach is to “purge” the channel with media or buffer,displacing the solution containing unselected cells from thepartitioning channel and into, e.g., a waste reservoir. Alternatively,the unselected cells can be displaced by the action of flowing thesolution comprising a plurality of individual cells (or cellular units)into the partitioning channel.

13. Cell Interactions

Based on the determined property, captured cells are independentlytransported to specified cell holding chambers. “Independentlytransported” means that each individual captured cell can be transferredto a specified destination chamber according to the needs of thepractitioner. Cells can therefore be transported so that thecharacteristics of each cell or combination of cells in each destinationchamber can are known. Typically (but optionally) cells are cultured inthe cell holding chambers. The effect of Interactions between individualcells (or cellular units) with known properties (e.g., of know celltypes), and/or their progeny, can be studied.

Tables 1 and 2 illustrate this by imagining a device with 4 Segments(1-4), 5 Cell holding chambers (1-5), and two cell types (A and B). Inthis hypothetical, after six rounds of cell capturing, determination andtransport are carried out, as summarized in Table 1, cells aredistributed to Cell holding chambers 1-5 are shown in Table 2.

TABLE 1 Round Cell Segment Destination Chamber (6 rounds) Property (4segments) (5 chambers) 1 A 4 1 1 A 3 3 2 B 4 2 3 A 1 3 4 B 2 3 4 B 3 4 5B 1 4 6 A 4 5

As illustrated in Table 2, after two days of culture a gene expressionpattern (e.g., expression of k-ras) can be determined for the cells ineach of the cell holding chambers. In the hypothetical results shown inTable 2, the effect of interactions of cell type A and cell type B isincreased expression (by one or both of the cell types) of the K-rasgene.

TABLE 2 Gene Expression Pattern Chamber Seed Cell(s) After 2 DaysCulture 1 A Low 2 B Low 3 A + B High 4 B + B Low 5 A + A Low

The number of cell holding chambers associated with a partitioningchannel (or pair of partitioning channels) can vary but is typically inthe range 1-150, more often 10-100, and often 35-75.

Other properties of cell holding chambers, and cell culture in them, arediscussed below.

14. Harvest

Cells may be cultured in the cell holding chambers for a desired periodof time (e.g., 1 hour to 4 weeks). Typically, short term culture is used(e.g., 1 hour to 24 hours). During culture cells may be observed and,optionally, cells may be manipulated or challenged. For example, cellsmay be contacted with microRNAs to effect transdifferentiation. In someembodiments the cells either captured or in culture can be assayed withcomponents that result in a change of extracellular or intracellulardetectable signals such as a colormetric or fluorescent molecule. At theend of the culture period, the cells may be discarded, recovered asviable cells, or lysed or otherwise made non-viable for analysis.

14.1. Cells Recovered as Viable Cells

Viable cells may be recovered from the cell-holding chambers in a numberof ways. In one approach the cell(s) are enzymatically detached from thesubstrate, if necessary, and then flowed out of the cell holding chamberfor collection. The flow path can be, in principal, the reverse of thepath by which cells were transported into the cell holding chamber.Alternatively, an auxiliary exit channel can be used.

In another approach the system is configured to be easily disassembledto expose the cell holding chambers, allowing the contents to be removed(e.g., using a micropipette). In one approach adherent cells are removedby moving the substrate to which the cells are adhered out of themicrofluidic system.

14.2. Analysis of Cell Components

In some embodiments, following a culture period, the cell(s) in a cellholding chamber are processed (e.g., lysed) to release cell componentssuch as nucleic acids, proteins and the like. With reference to FIG. 6,a lysis solution can be introduced through the main input or,alternatively, through an auxiliary channel (not shown). Cell componentscan then be carried into the “main drain” for collection and analysis.The ordinarily skilled practitioner guided by this disclosure will beable to design analogous structure when cell holding chambers withdifferent designs are used.

In some embodiments the cell component analysis is carried out in themicrofluidic system, such as in a multi-chamber reaction configurationfluidically connected to the cell holding chamber. Analysis includesnucleic acid analysis (e.g., amplification, sequencing or cloning of RNAor DNA from the cell(s)), protein analysis, cell fixation, and any othermethods for characterizing a cell or combination of cells. Forillustration and not limitation, FIG. 8 shows an example of amulti-chamber reaction configuration 220-c in accordance with variousembodiments. Multi-chamber reaction configuration 220-c may includedifferent components or aspects in accordance with various embodiments.Multi-chamber reaction configuration 220-c may be configured to performdifferent processes including, but not limited to, STA, RT-STA,mRNA-SEQ, preamplification, WMA, multimodal applications, proteinapplications, sample processor applications, WTA, WGA, real-time PCRpreparation, CNV, and/or haplotyping. Multi-chamber reactionconfiguration 220-c may be configured to perform multiple reactionsteps, which may include active mixing.

Multi-chamber reaction configuration 220-c may include numerous valves520, which may be utilized to control the flow of solutions throughmulti-chamber reaction configuration 220-c. In some embodiments, a pump530, such as a peristaltic pump, may be included in multi-chamberreaction configuration 220-c to facilitate transport of solutionsthrough multi-chamber reaction configuration 220-c. Pump 530 may includemultiple valves 520; in this example, pump 530 may include three valves.One or more pumps 530 may be located at different locations.

Multi-chamber reaction configuration 220-c may also include multiplereaction chambers 510. In some embodiments, capture configuration 210-cmay be considered one of the reaction chambers 510. Merely by way ofexample, valves 520-d, 520-e, 520-f, and 520-g may be utilized tocontrol the direct flow between reaction chambers 510-a, 510-b, 510-i,and 510-j respective. Additional valves such as valves 520-h-520-o, indifferent combinations, may be utilized to introduce reagents, mixand/or circulate solutions from one or more reaction chamber 510.Additional valves 520-a and/or 520-b may control flow between differentcapture configurations. Valve 520-b may be utilized to control flow ofsolutions such as reagents to capture configuration 210-c. Multi-chamberreaction configuration 220-c may be configured to mix and/or circulatesolution during thermal cycling. Reaction products may be delivered 540to export configuration (not shown) may be referred to as a harvestconfiguration, harvest well, and/or harvest inlet in some cases.

The multi-chamber reaction configuration is flexible and can be used toisolate and amplify nucleic acids, isolate proteins, make cDNA, and awide variety of other molecular biological process and assay steps. Thesystem may be also include additional fluidic circuits including, butnot limited to, various published or commercially available systems. Forexample, the Fluidigm BioMark™ HD System can be used for geneexpression, single-cell gene expression, single-cell mRNA sequencing,SNP genotyping, copy number variation, sample quantification forsequencing. Fluidigm's qdPCR 37K™ IFC chip may be used for digital PCR.These and other assay systems can be integrated into the system of theinvention (i.e., fluidically connected by microfluidic channels)allowing, among other advantages greater automation and shorterprocessing time. See U.S. Pat. No. 7,604,965; U.S. Pat. Pub. Nos.2012-0115143 (“Universal Probe Assay Methods”), US 2012-0288857(“Multifunctional Probe-Primers”), and US 2013-0045881 (“Probe BasedNucleic Acid Detection”); copending commonly owned International PatentApplication No. PCT/US2012/065376 (“Nucleic Acid Detection UsingProbes”), and copending commonly owned provisional application61/799,559 (“Simultaneous Detection Of Target Protein And Target NucleicAcids In A Single Cell”), each of which is expressly incorporated byreference for all purposes.

15. Imaging

In preferred embodiments the system includes an imaging module,integrated into the for imaging one or more cells and locations of themicrofluidic device. Methods and equipment for collecting and processingimages are known in the art. The imaging module may include at least amicroscope or a camera configured to image one or more captured cells inthe microfluidic device. In some embodiments the imaging modulecomprises a CCD camera.

The imaging module may be configured to image one, more than one, or allof the areas of the integrated fluidic circuit, including aspartitioning channel(s), cell holding chambers, partitioning channelsectors, holding chamber sectors, post-culture processing sectors, andassay (amplification) sectors.

In some embodiments (e.g., imaging a detectably labeled cell) it isnecessary to illuminate the cell(s) and/or reactions at an appropriatewave length, for example in detecting cells labeled with a fluorescenttag or, for example and not limitation, for analysis of an amplificationreaction in which a. fluorescent signal is generated. Elements such as afilter wheel and other optical elements are known to those of ordinaryskill in the art. See, for example, U.S. Pat. No. 7,906,072 incorporatedherein by reference.

In one embodiment substantially the entire microfluidic circuit isimaged and optionally displayed to the user. In other embodiments, thepartitioning channel is imaged and the cell holding chambers are imaged,without imaging manifolds. In some embodiments software-driven imageanalysis is used to monitor cells.

The imaging module may be a CCD or CMOS camera that is configured toimage the entire microfluidic device without a stitching or scanningprocess. In one embodiment, the imaging module is further configured toimage fluorescence emissions from reactions performed on or within themicrofluidic device after cells have been selected and captured. Theconfiguration for selecting the wavelength to be imaged may includefluorescent filters arranged in a filter wheel or a filter cubeconfiguration. In a further embodiment, the filtering of the fluorescentradiation is performed in the imaging module itself, such as within theimaging surface, or by software. The size of the imaged area includingcell capture and cell holding sites and reaction sites may be about30.5×30.5 mm (930.25 mm²) or greater. In another embodiment, the imagedarea including cell capture and cell holding sites and reaction siteswill be from about 930 to about 1200 mm² or from about 1200 to 1600 mm²or from about 1600 to 2000 mm². In a further embodiment, the imaged areawill be from about 2000 to 2600 mm². The imaging module will generallyhave an imaging surface, such as a CCD or CMOS imaging surface, which isequivalent to or larger than the imaged area. The imaging surface of theimaging module will typically have from 15,000,000 to 100,000,000pixels, and sometimes to 200,000,000 pixels, each about 6 to 9 micronsin size. In some embodiments, the pixels are about 9 microns in size. Auseful range for pixel size is from about 3 to 6 microns or in someembodiments, from about 5 to 7 microns, or from about 6 to 10 microns.In another embodiment, the pixels are about 6 microns in size. Theimaging area may further have from about 200,000,000 to about300,000,000 pixels or from about 300,000,000 to 400,000,000 pixels, Inanother embodiment, the imaging area will have from about 500,000,000pixels to about 750,000,000 pixels.

It will be appreciated that in embodiments in which additional assaysoccur in the system (e.g., real-time PCR assays) the imaging module maybe used both for observing the characteristics and movement of cells andfor detection of PCR and other reaction products.

16. Automation

16.1 Computer Implementation

It is contemplated that the methods of the invention will be partiallyautomated. For example, based on user input actuation of valves, controlpumps, movement of fluid and cells, and imaging can be implemented bycomputer. It is expected the system will include one or more computerprocessor modules and/or one or more memory modules that may be used.The computer functions can be integrated into the device or be remotefrom it.

16.2 User Interface

In one aspect the invention provides a user interface to easily carryout automated steps of the method. FIG. 9 provides a virtual screen shotof an exemplary graphical user interface. In general terms, atouchscreen device is provided which shows segments (images or agraphical representation of segments), captured cells in segments(images or a graphical representation of cells, optionally including arepresentation of cell characteristics) and cell holding chambers(images or a graphical representation of cell holding chambers). Imagesmay be produced using a CCD camera. In one embodiment the touchscreendevice shows images of cells and segments, and representations of cellholding chambers. In one embodiment the touchscreen device shows imagesof cells, and representations of segments and cell holding chambers. Theterm “representation” is used to encompass both images and graphicalrepresentations.

The user, having identified a captured cell or, equivalently, a segmentcontaining a captured cell, of interest instructs the system totransport the cell to a selected cell holding chamber (target). Avariety of user gestures can be used. For example, using a drag-and-dropgesture the use taps the image of the cell or segment and drags a fingeror cursor to the representation of s specified cell holding chamber.Optionally, an image of the cell moving from the segment to the cellholding chamber is shown. Other gestures may be used. For example, theuser may tap on the representation cell (object) at the first location(segment) and then tapping at the destination location (cell holdingchamber).

Accordingly, the invention provides a method for transporting cells bymanipulating objects in a graphical user interface for a computer, ofthe type in which representations of objects stored in a memory aredisplayed to a user on a display, comprising the steps of selecting afirst cell whose representation is displayed on said display; draggingthe representation of the first cell from a first segment location onthe display to a first cell holding chamber; and individually selectingeach of a plurality of additional cells and transporting each of them toindependently selected cell holding chambers which may the same as ordifferent from the first cell holding chamber Further, the inventionprovides a graphical user interface for a computer having a displaydevice, comprising a user-controlled component for selecting and movingrepresentations of cells displayed on said display device from a segmenton said display device to a cell holding chamber location displayed onsaid device.

17. Device

An exemplary system of the invention is illustrated in FIG. 10. FIG. 10Ashows a top oblique view of the microfluidic chip holding apparatus fromthe left side. FIG. 10B shows an exploded view of the apparatus from theright side. The holding apparatus is configured to operate themicrofluidic device by supplying reagents, maintaining the appropriatetemperature and humidity, and operating valves to control the flow offluid. It comprises a platform at the front side, and a mix box on theback side (shown on the left in FIG. 10A and in the right in FIG. 10B).

Referring to FIG. 10B, the base of the apparatus is an IFC carrier withsample input wells with a cavity shaped and sized to receive amicrotiter plate or chip: typically 35 mm square. Above the carrier isan interface plate comprising gaskets connected to control channels inthe chip that operate valves by pneumatic control. Above the plate is alayer of polyimide that is configured as an integrated heater tomaintain a desired temperature on the platform surrounding the chip, andis connected up to the mix box to bring the input gas mixture up totemperature. Above the heater on the platform is a glass surfacesurrounded by a glass bracket. The glass is iridium tin coated toinhibit condensation from the moist gasses supplied to the chip. Theglass surface is configured to be optically transparent so that eachcell in the chip can be located and characterized according to itsmorphology by imaging. Referring to FIG. 10A, the mix box receives airor a gas mixture appropriate for keeping cells viable in culture(typically 95% O2 and 5% CO2), which is fed from the mix box through theplatform to gas supply channels in the chip. The gas mixture is made atleast 60%, 80% or 90% humidity to inhibit loss of moisture from theholding chambers of the chip through gas supply channels. After passingthrough the chip, the humid gas mixture exits from the platform throughthe mix box and out the humid air exit tube, taking with it anycondensation that has formed near the chip.

18. Exemplary Valves, Detectable Labels, Cells, Microfluidic Systems

18.1: Valves

Valves of various types are known in the art, including micromechanicalvalves, elastomeric valves, solid-state microvalves, and others. See,e.g., Felton, 2003, The New Generation of Microvalves” AnalyticalChemistry 429-432. Two common approaches to fabrication ofmicroelectromechanical (MEMS) structures such as pumps and valves aresilicon-based bulk micro-machining (which is a subtractive fabricationmethod whereby single crystal silicon is lithographically patterned andthen etched to form three-dimensional structures), and surfacemicro-machining (which is an additive method where layers ofsemiconductor-type materials such as polysilicon, silicon nitride,silicon dioxide, and various metals are sequentially added and patternedto make three-dimensional structures).

In one embodiment, the valve is a monolithic valve. In a preferredembodiment the valve is a pressure-actuated “elastomeric valve.” Apressure-actuated elastomeric valve consists of a configuration in whichtwo microchannels are separated by an elastomeric segment that can bedeflected into or retracted from one of the channels (e.g., a flowchannel) in response to an actuation force applied to the other channel(e.g., a control channel). Examples of elastomeric valves includeupwardly-deflecting valves (see, e.g., US 20050072946), downwardlydeflecting valves (see, e.g., U.S. Pat. No. 6,408,878), side actuatedvalves (see, e.g., US 20020127736, e.g., paragraphs 0215-0219],normally-closed valves (see, e.g., U.S. Pat. Nos. 6,408,878 and6,899,137) and others. A chemical resistant microfluidic valve with anelastomeric component is described by Hua et al., 2006, J MicromechMicroeng 16:1433-1443. In some embodiments a device can have acombination of valves (e.g., upwardly and downwardly deflecting valves).Valves can be actuated by injecting gases (e.g., air, nitrogen, andargon), liquids (e.g., water, silicon oils and other oils), solutionscontaining salts and/or polymers (including but not limited topolyethylene glycol, glycerol and carbohydrates) and the like into thecontrol channel. Some valves can be actuated by applying a vacuum to thecontrol channel.

In addition to elastomeric valves actuated by pressure-based actuationsystems, monolithic valves with an elastomeric component andelectrostatic, magnetic, electrolytic and electrokinetic actuationsystems may be used. See, e.g., US 20020109114; US 20020127736, e.g.,0168-0176; and U.S. Pat. No. 6,767,706. One-way valves have also beendescribed (see, e.g., Adams et al., 2005, J. Micromech. Microeng.

Other microvalves include microvalve based on electromagnetic actuation(electromagnetic solenoid plungers), piezoelectric effect (disk,cantilever and stack types), pneumatic and thermopneumatic systems,electrostatic actuators, and bimetallic beams (see Shoji et al. 1994, J.Micromech. Microeng. 4(4): 157-171, December 1994), hydrogel-basedvalves that expand or contract (and thereby close or open) in responseto pH changes (Beebe et al. Nature 404(6778): 588-90, 6 Apr. 2000),microfluidic valves comprising electrochemically generated bubbles (Huaet al. Anal Chem. 74(24): 6392-6, 15 Dec. 2002), valves based on polymermonoliths, actuated by UV light (Hasselbrink et al. Anal Chem. 74(19):4913-8, 1 Oct. 2002), microvalves controlled by electrostatics [U.S.Pat. Nos. 5,417,235 and 5,452,878] or by the thermal buckling ofmaterials (U.S. Pat. No. 5,785,295).

18.3: Detectable Labels

For monitoring, evaluating, imaging, and otherwise processing a cell byoptical means, a suitable detectable label may be, for example and notlimitation, colored, fluorescent, luminescent, or phosphorescent. It maybe conjugated or bound to the cell surface, or incorporated inside thecell. In some embodiments, it constitutes or is conjugated to anantibody, a lectin, a ligand, a substrate, or a reaction product.Individual cells (or cellular units) may also be tracked and monitoredaccording to a morphological feature that distinguishes them from othercells being processed in the device.

18.4: Cells

The microfluidic devices of this invention typically have channels andchambers that are sized and shaped to permit passage of eukaryotic cellsthat are both diploid and nucleated. Without implying any limitation onthe practice of the invention, such a cell may be derived from avertebrate, a mammal, a domesticated animal, a mouse, a primate, or ahuman. It may be an astrocyte, a neuron, a Schwann cell, an epithelialcell, an endothelial cell, an adipocyte, a renal cell, an exocrine cell,a fibroblast, a chondrocyte, an odontocyte, an islet cell, a cardiaccell, a smooth muscle cell, a striated muscle cell, a renal cell, ahepatocyte, a Kuppfer cell, a pituitary cell, a mucous cell, a hormonesecreting cell, a keratinocyte, a basal cell, a pneumal cell, apericyte, or a pulposus cell. It may be a blood related cell such as anerythrocyte, a reticulocyte, a megakaryocyte, a monocyte, a macrophage,a dendritic cell, a granulocyte, an eosinophil, a neutrophil, abasophil, a mast cell, a lymphocyte, a cytotoxic T cell, a helper Tcell, a suppressor T cell, a B lymphocyte, a natural killer cell, or adendritic cell. It may be a cancer or cancer stem cell of various kinds,a leukemia or lymphoma cell, or a hybridoma. It may be a pluripotent ortissue-specific stem cell, a progenitor cell, an oocyte, or a germ cell.Alternatively, it may be a plant cell such as a parenchymal cell, acollenchymal cell, a Sclerenchymal cell, a sclerid cell, a meristematiccell, a xylem cell, or an epidermal cell. Alternatively, it may be aunicellular organism such as a protist or a fungi such as yeast. Cellsthat are processed or cultured together may be the same type or aplurality of different types in any combination.

Also contemplated is the processing of haploid cells, prokaryotes suchas bacteria, anucleated cells such as platelets, and a non-livingparticles and other entities. In some embodiments, the microfluidicdevice and its channels are scaled and configured so as to process andallow passage of such cells and particles individually. In otherembodiments, the microfluidic device and its channels are scaled andconfigured so as to process such cells or particles in bulk.

In some embodiments, the cell: is not a gamete, is not an ovum, is not asperm.

In some embodiments, the combination of cells cultured in a cell holdingchamber does not comprise gametes.

In some embodiments, the combination of cells cultured in a cell holdingchamber does not comprise both an ovum and a sperm.

18.5: Microfluidic Systems

Devices of the invention can be constructed out of any of variousmaterials or combination of materials from channels, valves and othermicrofluidic components can be fabricated. Materials from which a chipcan be fabricated include, without limitation, elastomers, silicon,glass, metal, polymer, ceramic, inorganic materials, and/or combinationsof these materials.

The methods used in fabrication of a microfluidic device will vary withthe materials used, and include soft lithography methods, microassembly,bulk micromachining methods, surface micro-machining methods, standardlithographic methods, wet etching, reactive ion etching, plasma etching,stereolithography and laser chemical three-dimensional writing methods,modular assembly methods, replica molding methods, injection moldingmethods, hot molding methods, laser ablation methods, combinations ofmethods, and other methods known in the art or developed in the future.A variety of exemplary fabrication methods are described in Fiorini andChiu, 2005, “Disposable microfluidic devices: fabrication, function, andapplication” Biotechniques 38:429-46; Beebe et al., 2000, “Microfluidictectonics: a comprehensive construction platform for microfluidicsystems.” Proc. Natl. Acad. Sci. USA 97:13488-13493; Rossier et al.,2002, “Plasma etched polymer microelectrochemical systems” Lab Chip2:145-150; Becker et al., 2002, “Polymer microfluidic devices” Talanta56:267-287; Becker et al., 2000, “Polymer microfabrication methods formicrofluidic analytical applications” Electrophoresis 21:12-26; U.S.Pat. No. 6,767,706 B2, e.g., Section 6.8 “Microfabrication of a SiliconDevice”; Terry et al., 1979, A Gas Chromatography Air AnalyzerFabricated on a Silicon Wafer, IEEE Trans. on Electron Devices, v.ED-26, pp. 1880-1886; Berg et al., 1994, Micro Total Analysis Systems,New York, Kluwer; Webster et al., 1996, Monolithic Capillary GelElectrophoresis Stage with On-Chip Detector in International ConferenceOn Micro Electromechanical Systems, MEMS 96, pp. 491496; and Mastrangeloet al., 1989, Vacuum-Sealed Silicon Micromachined Incandescent LightSource, in Intl. Electron Devices Meeting, IDEM 89, pp. 503-506.

In some embodiments, the device is fabricated using elastomericmaterials. Fabrication methods using elastomeric materials will only bebriefly described here, because elastomeric materials, methods offabrication of devices made using such materials, and methods for designof devices and their components have been described in detail (see,e.g., Unger et al., 2000, Science 288:113-16; U.S. Pat. No. 6,960,437(Nucleic acid amplification utilizing microfluidic devices); U.S. Pat.No. 6,899,137 (Microfabricated elastomeric valve and pump systems); U.S.Pat. No. 6,767,706 (Integrated active flux microfluidic devices andmethods); U.S. Pat. No. 6,752,922 (Microfluidic chromatography); U.S.Pat. No. 6,408,878 (Microfabricated elastomeric valve and pump systems);U.S. Pat. No. 6,645,432 (Microfluidic systems includingthree-dimensionally arrayed channel networks); U.S. Patent Applicationpublication Nos. 2004/0115838, 20050072946; 20050000900; 20020127736;20020109114; 20040115838; 20030138829; 20020164816; 20020127736; and20020109114; PCT patent publications WO 2005/084191; WO05030822A2; andWO 01/01025; Quake and Scherer, 2000, “From micro to nanofabricationwith soft materials” Science 290: 1536-40; Xia et al., 1998, “Softlithography” Angewandte Chemie-International Edition 37:551-575; Ungeret al., 2000, “Monolithic microfabricated valves and pumps by multilayersoft lithography” Science 288:113-116; Thorsen et al., 2002,“Microfluidic large-scale integration” Science 298:580-584; Chou et al.,2000, “Microfabricated Rotary Pump” Biomedical Microdevices 3:323-330;Liu et al., 2003, “Solving the “world-to-chip” interface problem with amicrofluidic matrix” Analytical Chemistry 75, 4718-23,” Hong et al,2004, “A nanoliter-scale nucleic acid processor with parallelarchitecture” Nature Biotechnology 22:435-39; Fiorini and Chiu, 2005,“Disposable microfluidic devices: fabrication, function, andapplication” Biotechniques 38:429-46; Beebe et al., 2000, “Microfluidictectonics: a comprehensive construction platform for microfluidicsystems.” Proc. Natl. Acad. Sci. USA 97:13488-13493; Rolland et al.,2004, “Solvent-resistant photocurable “liquid Teflon” for microfluidicdevice fabrication” J. Amer. Chem. Soc. 126:2322-2323; Rossier et al.,2002, “Plasma etched polymer microelectrochemical systems” Lab Chip2:145-150; Becker et al., 2002, “Polymer microfluidic devices” Talanta56:267-287; Becker et al., 2000, “Polymer microfabrication methods formicrofluidic analytical applications” Electrophoresis 21:12-26; Terry etal., 1979, A Gas Chromatography Air Analyzer Fabricated on a SiliconWafer, IEEE Trans. on Electron Devices, v. ED-26, pp. 1880-1886; Berg etal., 1994, Micro Total Analysis Systems, New York, Kluwer; Webster etal., 1996, Monolithic Capillary Gel Electrophoresis Stage with On-ChipDetector in International Conference On Micro Electromechanical Systems,MEMS 96, pp. 491496; and Mastrangelo et al., 1989, Vacuum-Sealed SiliconMicromachined Incandescent Light Source, in Intl. Electron DevicesMeeting, IDEM 89, pp. 503-506; and other references cited herein andfound in the scientific and patent literature. In general, the differentmicrofluidic devices described herein may fabricated utilizing a varietyof fabrication methods using elastomeric materials, such as PDMS, andmethods for design of the microfluidic devices and their components havebeen described in detail in the scientific and patent literature. See,e.g., Unger et al. (2000) Science 288:113-116; U.S. Pat. No. 6,960,437(Nucleic acid amplification utilizing microfluidic devices); U.S. Pat.No. 6,899,137 (Microfabricated elastomeric valve and pump systems); U.S.Pat. No. 6,767,706 (Integrated active flux microfluidic devices andmethods); U.S. Pat. No. 6,752,922 (Microfluidic chromatography); U.S.Pat. No. 6,408,878 (Microfabricated elastomeric valve and pump systems);U.S. Pat. No. 6,645,432 (Micro fluidic devices includingthree-dimensionally arrayed channel networks); U.S. Patent ApplicationPublication Nos. 2004/0115838; 2005/0072946; 2005/0000900; 2002/0127736;2002/0109114; 2004/0115838; 2003/0138829; 200210164816; 2002/0127736;and 2002/0109114; PCT Publication Nos. WO 2005/084191; WO 05/030822A2;and WO 01101025; Quake and Scherer, 2000, “From micro to nanofabricationwith soft materials” Science 290: 1536-40; Unger et at., 2000,“Monolithic microfabricated valves and pumps by multilayer softlithography” Science 288: 113-116; Thorsen et at., 2002, “Micro fluidiclarge-scale integration” Science 298:580-584; Chou et at., 2000,“Microfabricated Rotary Pump” Biomedical Microdevices 3:323-330; Liu etat., 2003, “Solving the “world-to-chip” interface problem with amicrofluidic matrix” Analytical Chemistry 75, 4718-23, Hong et. al,2004, “A nanoliter-scale nucleic acid processor with parallelarchitecture” Nature Biotechnology 22:435-39, all incorporated byreference for all purposes.

Certain characteristics of microfluidic channels will vary with theirfunction. For example, channels through which cells are transported willhave dimensions suitable for the types of cells being studied. Channelsthough which solutions (e.g., cell media) are transported typically havesmaller dimensions. Control channels, which are used in certainelastomeric microfluidic devices to actuate valves, may have dimensionsthat vary. Thus the dimensions of channels can vary widely but typicallyinclude at least one cross-sectional dimension (e.g., height, width, ordiameter) less than 2 mm, generally less than 1 mm, sometimes less than0.5 mm, and sometimes less than 0.3 mm. Channels often have at least onecross-sectional dimension in the range of 0.05 to 1000 microns,sometimes 0.2 to 500 microns, and sometimes 10 to 250 microns. Thechannel may have any suitable cross-sectional shape that allows forfluid and/or cell transport, for example, a square channel, a circularchannel, a rounded channel, a rectangular channel, etc. In an exemplaryaspect, flow channels are rectangular and have widths of about in therange of 0.05 to 1000 microns, sometimes 0.2 to 500 microns, andsometimes 10 to 250 microns. In an exemplary aspect, channels havedepths of 0.01 to 1000 microns, sometimes 0.05 to 500 microns, sometimes0.2 to 250 microns, and sometimes 1 to 100 microns. In an exemplaryaspect, flow channels have width-to-depth ratios of about 0.1:1 to100:1, more preferably 1:1 to 50:1, more preferably 2:1 to 20:1, andmost preferably 3:1 to 15:1, and often about 10:1.

Microfluidic devices of the present invention may include one or moreintegral pumps for transport of fluids through flow channels and intoand out of other device components (e.g., column or reactors) or thedevice itself. Suitable pumps can be electronic, electrostatic,magnetic, mechanical, syringe, pneumatic, or peristaltic. Preferablyperistaltic pumps, such as those described in U.S. Pat. No. 6,408,878are used. Alternatively pumps can be external to the chip. Pumps arealso used to transport fluids (e.g., water) through control channels toactuate valves. Pumps are also used to draw a vacuum in, for example,vent channels.

Other microfluidic components also can be integrated into the chip,including, for example, control channels, guard channels, vent channels,fluid reservoirs, mixing reactors, rotary mixers, separation modules(e.g., separation columns), sorting regions, pumps, ports, vias,nozzles, monitoring systems, lenses, sensors, temperature controlsystems, heat sources, light sources, waveguides and the like.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. In addition, all other publications,patents, and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

1.-52. (canceled)
 53. A chamber in a microfluidic device that isconfigured for receiving and maintaining live cells, the chambercomprising: an input channel configured so that nucleated eukaryoticcells can pass intact through the input channel and into the chamber; aplurality of four or more drain channels configured so that fluid butnot nucleated eukaryotic cells can exit the chamber; and a gas permeablemembrane separating the chamber from a supply channel, wherein thesupply channel is configured to bring a gaseous mixture to the chamber,and the gas permeable membrane is configured so that gasses may beexchanged between the chamber and the supply channel.
 54. The chamber ofclaim 53, wherein the chamber or the input channel is connected to areagent channel configured for supplying a fluid comprising one or morereagents for maintaining or treating cells in the chamber.
 55. Thechamber of claim 54, wherein the reagent channel is connected to asupply of a fluid containing cell nutrients.
 56. The chamber of claim54, wherein the reagent channel is connected to a supply of a fluid thatlyses cells upon delivery of the fluid into the chamber.
 57. The chamberof claim 53, wherein the input channel tapers towards the chamber,thereby directing cells towards the chamber's center.
 58. The chamber ofclaim 53, wherein the drain channels are distributed around the chamberin a manner that allows fluid to flow from the input channel into thechamber and out the drain channels without drawing cells in the chambertowards the drain channels.
 59. The chamber of claim 58, wherein thechamber has a perimeter that is substantially circular or oval in shape,and the drain channels are distributed over more than 180 degrees of theperimeter.
 60. (canceled)
 61. The chamber of claim 53, wherein the drainchannels connect to the chamber through drain openings and the inputchannel connects to the channel though an input opening, and thediameter of the drain openings is less than 20% of the diameter of theinput opening, thereby inhibiting passage of intact cells into the drainchannels.
 62. The chamber of claim 53, wherein the drain channelscontain beads or a filter, thereby inhibiting passage of intact cellsinto the drain channels. 63.-64. (canceled)
 65. The chamber of claim 53,at least 50 microns across at its narrowest diameter.
 66. (canceled) 67.The chamber of claim 53, comprising a convex lower surface.
 68. Thechamber of claim 53, comprising a lower surface coated with a substancethat promotes cell adhesion.
 69. The chamber of claim 68, wherein thesubstance is an extracellular matrix.
 70. The chamber of claim 68,wherein the substance is fibronectin.
 71. The chamber of claim 53,wherein the chamber, the input channel, and the drain channels arefilled with fluid.
 72. The chamber of claim 53, containing at least twoeukaryotic cells.
 73. A plurality of four or more chambers according toclaim 53, each having its own separate input channel, wherein each ofthe input channels is configured to supply cells from a common source.74.-76. (canceled)
 77. A method of maintaining cells in culture,comprising delivering cells to a chamber according to claim 53,supplying nutrient medium into the chamber through the input channel ora separate reagent channel, and supplying gas to the gas permeablemembrane through the supply channel.
 78. The method of claim 77, furthercomprising individually selecting the cells delivered to the chamberfrom a cell mixture.
 79. A method of extracting intracellular componentsfrom one or more cells that are present in a chamber according to claim53, comprising lysing the cells by delivering a fluid that causes celllysis into the chamber, and retrieving products of the lysis from thechamber.
 80. The method of claim 79, wherein the products are retrievedthrough the drain channels.
 81. The method of claim 79, wherein theproducts comprise nucleic acid.
 82. The method of claim 79, furthercomprising subjecting the products to a chemical or biochemicalreaction. 83.-124. (canceled)